We propose (1) to delineate the enzymatic and/or cofactor binding characteristics of each of the peptide components of a mitochondrial membrane fraction (complex I), (2) to evaluate the orientation (i.e., spacial relationship) amongst the peptides of this enzyme complex, and (3) to identify the active site region for the adenine and nicotinamide components of NADH on a purified protein from complex I (i.e., the NADH-dehydrogenase). (4) In addition, the FMN binding region on the NADH-dehydrogenase and its relationship to the NADH binding region will be identified. The principal method of approach is the use of a series of arylazido pyridine, adenine and flavin nucleotide photoaffinity analogues developed in this laboratory as general reagents for the investigation of the nucleotide binding regions of proteins. The application of these probes to the study of complex I shall be combined with gel electrophoresis in sodium dodecylsulfate and studies using cross-linking reagents. The analysis of the active site region for cofactor binding on NADH-dehydrogenase shall use the standard techniques of peptide isolation, purification, and sequence analysis applied to the protoprobe-labeled purified dehydrogenase.